<p>Wheat yellow mosaic virus (WYMV), a member of the genus <i>Bymovirus</i>, causes substantial losses in wheat production in East Asia, including China and Japan. Although genomic RNA replication is an important process in the viral life cycle, the independent regulation of WYMV RNA replication by its 3ʹ and 5ʹ untranslated regions (UTRs) remains unclear. In this study, we sought to analyze the core structural features of the 3ʹ UTR in the regulation of the replication of WYMV RNA1 in vitro, locate the viral RNA sites to which the NIb protein binds, and determine the regulatory effects of the VPg, P3, and 14&#xa0;K proteins on NIb RNA-dependent RNA polymerase (RdRp) activity. We found that the NIb protein in WYMV RNA1 only specifically recognized its 3ʹ UTR as the core enzyme for replication. Moreover, the 14&#xa0;K and P3 proteins were established to synergistically enhanc the in vitro RdRp activity of the NIb protein, whereas the VPg protein was found to play an inhibitory role. Based on RNA structure probing and mutational analysis, we identified ⁷⁶²⁴UU and ⁷⁵⁷¹UU as putative sites for interaction with the NIb protein and demonstrated that replication is dependent on their coexistence. In addition, hairpins 2 and 5 of the 3' UTR were found to be essential for NIb protein replication. Collectively, the findings of our in vitro analysis of the replication regulatory elements of WYMV RNA1 3ʹ UTR, provide a basis for in vivo studies on the regulation of WYMV replication and the identification of potential targets for the prevention and control of WYMV-caused crop diseases.</p>